DETERMINATION OF ETHYL GLUCURONIDE IN HAIR SAMPLES

Ethyl glucuronide (EtG) is a non-volatile, water-soluble, stable-upon-storage, direct metabolite of ethanol and can be detected in body fluids and tissues (and also in post-mortem material) for an extended time period after the complete elimination of alcohol from the body (Alt et al., 1997; Schmitt et al., 1997; Seidl et al., 1998; Wurst et al., 1999a,b). The aim of the present Letter is to emphasize through our preliminary results that the detection of alcohol consumption that has taken place several weeks or months earlier, via the determination of EtG in hair, could be a useful forensic tool as well as for monitoring alcohol consumption of those in treatment for alcohol dependence.

Thirty-one hair samples from four groups of subjects were analysed: (1) hair was taken post mortem from 16 subjects with a known history of alcoholism; the diagnosis was established by police investigations including information from the subjects' family practitioners; (2) hair from four alcoholics (diagnosed according to ICD-10), who were hospitalized at the Alcohol and Drug Unit, Department of Psychiatry II, University of Ulm, Germany; (3) hair from six social drinkers with a self-reported alcohol consumption of up to 20 g/day; (4) hair from five children.

The proximal 2–8-cm hair segment was used for analysis. The hair samples were first washed with methanol and acetone (10 ml, 5 min) and were then cut into small pieces with an approximate length of 1 mm without any subdivision into segments. To a total of 100 mg of the cut hair samples, 100 ng deuterium-labelled (d5)-EtG (Medichem, Stuttgart, Germany) as internal standard and 2 ml demineralized water were added. After ultrasonication for 2 h at 30°C, the supernatants were separated after centrifugation (3000 rpm for 5 min), transferred into gas chromatograph vials and evaporated at 30°C under nitrogen. Following evaporation, 50 μl pyridine (Merck) and 100 μl BSTFA (N,O-bis(trimethylsilyl)-trifluoroacetamide; Merck) were added. The samples were vortex mixed and then placed in an incubator at 90°C for 30 min. After evaporation, the residues were dissolved in 50 μl ethyl acetate (Merck) for gas chromatography/mass spectrometry (GC/MS) analysis.

GC/MS analysis was performed on a Hewlett Packard 6890 gas chromatograph interfaced with a Hewlett Packard 5973 mass selective detector. The gas chromatograph was equipped with a 30 m × 0.25 mm (0.25 μm film thickness) HP-5MS (Hewlett Packard) column operated in the splitless mode with valve closed for 0.5 min. The carrier gas was helium. Injector and detector temperatures were 250°C and 280°C, respectively. Oven temperature was set for 100°C for 2 min, then increased at a rate of 15°C/min to 250°C and, finally, at 20°C/min to 280°C, which was maintained for 3 min. The total run time was 17.5 min. Data were acquired in the SIM-mode. The TMS-derivatives were identified with the following masses: m/z 160, 261, and 405 (target ion) for EtG and m/z 165, 266, and 410 for TMS-d5-EtG. For quantitative evaluation of EtG in hair samples, calibration graphs were created by adding EtG concentrations between 50 ng and 300 ng to the hair samples from the children.

In 18 out of 31 hair samples analysed, EtG was detected. The characterization of the hair samples as well as the results are detailed in Table 1⇔.

Measurable concentrations of EtG were not detected in the hair from six social drinkers with a daily consumption of up to 20 g ethyl alcohol or in the hair from the five children.

The analysis of EtG in hair might help judge a claimed longer period of abstinence. It remains to be elucidated why EtG could not be detected in two cases (subjects 14 and 26) despite a known history of alcohol misuse. The colour of hair as well as the use of hair colourings could be of importance in this issue. In cases where EtG is not detected alcohol consumption cannot be excluded. However, if the analysis of EtG in hair is positive, alcohol consumption has to be strongly assumed. In interpreting the results, external sources for EtG, e.g. sweat, have to be taken into consideration in every case. It remains to be shown whether or not a correlation between the amount of alcohol consumed and the EtG concentration in hair can be established. Our preliminary data indicate that it might be possible to discriminate social drinkers (up to 20 g/day) from alcoholics and to monitor abstinence by the determination of EtG in hair.